Stable liquid formulations of cyclophosphamide and processes to prepare the same

ABSTRACT

The present invention relates to stable liquid pharmaceutical formulations of cyclophosphamide comprising cyclophosphamide and at least one pharmaceutically acceptable excipient wherein moisture content of the liquid formulation is less than about 2.0% by weight. The invention further relates to stable liquid formulations of cyclophosphamide prepared by a process comprising a step of reducing the moisture content from cyclophosphamide or liquid compositions of cyclophosphamide or both. The invention further relate to method of using such stable liquid formulations of cyclophosphamide for parenteral administration either as ready-to-use or ready-to-dilute for treating various cancer disorders.

This application is a Continuation-in-Part of PCT InternationalApplication No. PCT/IB2015/055285, filed Jul. 13, 2015, which claims thebenefit of Indian Provisional Application Nos. 3454/CHE/2014, filed Jul.11, 2014, and 5215/CHE/2014, filed Oct. 17, 2014, all of which arehereby incorporated by references in their entireties.

FIELD OF THE INVENTION

The present invention relates to stable liquid formulations ofcyclophosphamide and processes to prepare the stable liquid formulationsof cyclophosphamide. The present invention relates to stablepharmaceutical liquid formulations of cyclophosphamide comprisingcyclophosphamide and at least one pharmaceutically acceptable excipientwherein moisture content of the liquid formulation is less than about2.0% by weight. The invention further relates to stable liquidformulations of cyclophosphamide prepared by a process comprising a stepof reducing the moisture content from cyclophosphamide or liquidcompositions of cyclophosphamide or both. The invention further relateto method of using such stable liquid formulations of cyclophosphamidefor parenteral administration either as ready-to-use or ready-to-dilutefor treating various cancer disorders.

BACKGROUND OF THE INVENTION

Cyclophosphamide is alkylating agent indicated for treatment of a)Malignant Diseases, Hodgkin's disease, lymphocytic lymphoma, mixed-celltype lymphoma, histiocytic lymphoma, Burkitt's lymphoma; multiplemyeloma, leukemias, mycosis fungoides, neuroblastoma, adenocarcinoma ofovary, retinoblastoma and breast carcinoma. b) Minimal Change NephroticSyndrome in Pediatric Patients (Only Oral dose is recommended).

Cyclophosphamide is a white crystalline powder with the molecularformula C₇H₁₅Cl₂N₂O₂P.H₂O and a molecular weight of 279.1.Cyclophosphamide is soluble in water, saline, or ethanol. The chemicalname for cyclophosphamide is2-[bis(2-chloroethyl)amino]tetrahydro-2H-1,3,2-oxazaphosphorine 2-oxidemonohydrate, The structure of cyclophosphamide monohydrate isrepresented by structure I.

The cytotoxic action of nitrogen mustard is closely related to thereactivity of the 2-chloroethyl groups attached to the central nitrogenatom. Under physiological conditions, nitrogen mustards undergointramolecular cyclization through elimination of chloride to form acyclic aziridinium (ethyleneiminium) cation. This highly unstable cationis readily attacked on one of the carbon atoms of the three memberaziridine ring by several nucleophiles, such as DNA guanine residues.This reaction releases the nitrogen of the alkylating agent and makes itavailable to react with the second 2-chloroethyl side chain, forming asecond covalent linkage with another nucleophile, thus interfering withDNA replication by forming intrastrand and interstrand DNA crosslinks.

As on today, two forms exist for cyclophosphamide i.e. cyclophosphamidemonohydrate form and anhydrous form. Cyclophosphamide monohydrate formis preferred for pharmaceutical processing, as the anhydrous form ishighly unstable and readily picks up water to form the monohydrate whenexposed to a relative humidity of about 20-30% or higher at about 25° C.

U.S. Pat. No. 3,018,302 disclose the cyclophosphamide as one of thenovel cyclic phosphoric acid ester amides.

U.S. Pat. No. 4,775,533 cover the method for reconstituting dry fillcyclophosphamide for use in injections without having to resort to theexpense of providing lyophilized products.

U.S. Pat. No. 5,036,060 discloses a stable lyophilizate ofcyclophosphamide without the use of mannitol, by using the sodiumchloride as the excipient.

U.S. Pat. No. 4,537,883 cover the lyophilized cyclophosphamidecompositions for reconstitution with water to provide a solution fororal or parenteral administration.

US20070265213A1: Covers composition for treating metastatic breastcancer and ovarian cancer, wherein composition comprisescyclophosphamide, docetaxel, doxorubicin.

U.S. Pat. No. 8,399,434 cover compositions of cyclophosphamide withmetalloporphyrin, wherein metalloprophyrin is included in an amount toenhance efficacy of cyclophosphamide.

As on today there are only solid formulations commercially available forcyclophosphamide. It is reported in the literature that nitrogenmustards exhibit poor stability in the aqueous solutions due to rapiddegradation, this is further supported that as on today there are noliquid formulations commercially available. Cyclophosphamide is nitrogenmustard belonging to the chemical class of Oxazaphosphorins. Baxter hasdeveloped and launched Cytoxan (cyclophosphamide for injection USP). Itis available as dry powder or lyophilized powders which onreconstitution with water are to be used immediately and whenreconstituted with 0.9% sodium chloride injection it is stable up to 24hours at room temperature and up to 6 days when refrigerated. Thereconstituted solutions upon further dilution with sodium 0.45% sodiumchloride injection it is stable up to 24 hours at room temperature andup to 6 days when refrigerated. Further when diluted with 5% dextroseinjection or combination with 5% dextrose+0.9% sodium chloride injectionit is stable up to 24 hours at room temperature and up to 36 hours whenrefrigerated. If it is reconstituted with water, the reconstitutedsolution is to be used immediately. Thus reconstituted and dilutedsolutions are stable only for a short period of time both at roomtemperature and refrigerated conditions and to be used within givenshorter timelines.

Further, the nursing personnel must be aware of occupational exposure ofpotentially carcinogenic cytostatic agents during the preparation ofreconstituted solutions, and contamination of the nursing staff shouldtherefore be avoided as much as possible. As a dry powder orlyophilizate, the drug must be dissolved prior to removal for injectionand then administered. Thus double handling of the drug. Thisnecessitates additional entry to the vial with a syringe to add thesolubilizing liquid vehicle. With each accession of the vial smallquantities of the drug become airborne and this is known asaerosolization. Such added exposure requires particular precautions suchas rubber gloves and masks. The Martindale Extra Pharmacopoeia. 28Editions. Page 175, left column, report about this, the adverse effectsof antineoplastic agents and recommends to handle these substances withgreat care and avoid contact with skin, eyes and not to be inhaled. Butit has been reported that in during preparation of solutions of drymatter (sterile crystallizate, lyophilizate), however, an inhalation ofsuch particles cannot be excluded with certainty. Furthermore,reconstitution introduces potential for dilution errors and may benecessary in some cases for a longer shaking period is required forsolubilizing the drug completely.

Further the most common desirable solvent for lyophilization is water.The reactivity of most nitrogen mustards in aqueous solutions presents achallenge to the industrial scale manufacture of lyophilized products.Thus the manufacturing and administering solid forms of injectable drugspresents several problems. In addition to that, the lyophilizationprocess is complex, costly and time consuming.

For the foregoing reasons, there is the need for “ready to use” or“ready to dilute” liquid formulations of cyclophosphamide. But formaking such formulations of cyclophosphamide, suitable solvent systemneed to be identified in which cyclophosphamide does not degrade andremains stable during its shelf life.

There are prior art documents disclosing the liquid formulations ofcyclophosphamide.

U.S. Pat. No. 4,952,575 cover storable solution of cyclophosphamide with80-100% v/v of ethanol at a temperature from about 15° C. to about 40°C. These reported ethanolic or ethanolic-aqueous liquid formulations arehighly concentrated with drug 10-70% w/v and ethanol aqueous solutions80-100% v/v. The patent also discloses that trials carried out withsolvents such as glycofurol, polyethylene glycol 300, polyethyleneglycol 400, 1,2 propylene glycol, 1,3 butylene glycol.

U.S. Pat. No. 4,879,286 cover cyclophosphamide liquid formulationcomprising 50 to 100% of an organic polyol and from about 0-50% water,particularly 80 percent propylene glycol and 20% polyethylene glycolgiving greatest stability for the dissolved cyclophosphamide.

US20050272698A1 cover aqueous composition of oxazaphosphorin such ascyclophosphamide, Mesna and etherified ß-cyclodex.

EP1023075B1 describes liquid compositions of oxazaphosphorin with thechloride ion source, wherein chloride ion stabilize the oxazaphosphorinin aqueous solutions.

US20140005148A1 cover compositions of cyclophosphamide with polyethylene glycol or propylene glycol or Glycerin or Dimethyl acetamide,polysorbate, polyethoxylated castor oil or combinations thereof. Furtherthe application discloses that solvents like ethanol or other polarprotic solvents are capable of nucleophilic attack on the carboncontaining the chlorine atom in mustard moiety which will lead toformation of degradation products.

One of the commonly used techniques to avoid such instability in aqueousenvironment is use of non-aqueous solvent system for preparing theformulation. But it has been observed that some of the water sensitivedrugs or excipients themselves have bound moisture causing instability.Due to this bound moisture the water sensitive drugs show instabilityeven in the presence of non-aqueous solvent system. Hence these type ofdrugs possess challenge to the formulation scientist when preparing theformulation even in the non-aqueous solvent system. Thus it is importantto remove the bound moisture or water content from the drug as well asfrom the excipients if required.

Even if we reduce water content from the drug or excipients individuallythere is high chance that during further process of preparing theformulation these drugs or excipients is exposed to environment and maypick up moisture. Hence removal of water content from drug or its liquidcompositions and maintaining the water content throughout the process iscritical in preparing the stable formulation.

However none of the above references disclose a stable formulations orcompositions of cyclophosphamide which are stable for longer period oftime.

Hence there is a still a need for the stable liquid formulations ofcyclophosphamide and the processes to prepare such stable liquidformulations of cyclophosphamide. None of the above disclosed documentsrefer to the stable compositions and process to prepare the stableliquid formulations of water sensitive drugs such as cyclophosphamidewhich can meet the requirements for the commercial use. This need isfulfilled by the present invention.

The instant invention has resulted from work undertaken to ascertain ifthe stability of cyclophosphamide in solution can be improved therebyallowing the marketing of such formulations and obviating theaforementioned shortcomings of currently available dry powder mixturesand lyophilized compositions. In addition, the processes of the presentinvention used to prepare the stable liquid formulations ofcyclophosphamide are simple, easy, cost effective, reproducible andquick to prepare.

SUMMARY OF THE INVENTION

The present invention relates to stable liquid formulations ofcyclophosphamide processes to prepare such stable liquid formulations ofcyclophosphamide. The invention relates to stable pharmaceutical liquidformulations of cyclophosphamide comprising cyclophosphamide and atleast one pharmaceutically acceptable excipient wherein moisture contentof the liquid formulation is less than about 2.0% by weight. Theinvention also relates to stable liquid formulations of cyclophosphamideprepared by a process comprising a step of reducing the moisture contentfrom cyclophosphamide or liquid compositions of cyclophosphamide orboth. The invention further relate to method of using such stable liquidformulations of cyclophosphamide for treating various cancer disorders.

In one of the embodiment the invention includes stable liquidformulations of cyclophosphamide comprising cyclophosphamide and atleast one pharmaceutically acceptable excipient.

In a embodiment the invention relates to stable liquid formulations ofcyclophosphamide comprising cyclophosphamide in pharmaceuticallyeffective concentration and at least one pharmaceutically acceptableexcipient.

In a embodiment the invention covers stable liquid formulations ofcyclophosphamide wherein pharmaceutically effective concentration ofcyclophosphamide is at least 0.1 g per mL

In an embodiment the invention covers stable liquid formulations ofcyclophosphamide wherein pharmaceutically effective concentration ofcyclophosphamide is at least 0.5 g per mL

In an embodiment the invention includes stable liquid formulations ofcyclophosphamide prepared by a process comprising a step of reducing themoisture content from cyclophosphamide.

In another embodiment the invention includes process for preparing thestable liquid formulation of cyclophosphamide wherein process comprisingthe steps of

-   -   a) reducing water content of cyclophosphamide by suitable drying        methods.    -   b) preparing the bulk solution of cyclophosphamide by dissolving        cyclophosphamide of step-a) in a suitable solvent.    -   c) filling bulk solution of step-b) in vials followed by        stoppering and sealing.

In an embodiment the invention includes stable liquid formulations ofcyclophosphamide wherein the moisture content of cyclophosphamide isreduced by selecting a suitable drying process selected from the groupcomprising of vacuum drying, lyophilization, solvent evaporation.

In one of the embodiment stable liquid formulations of cyclophosphamideprepared by removing the moisture content from cyclophosphamide isremoved by vacuum drying.

In another embodiment the invention includes stable liquid formulationsof cyclophosphamide prepared by a process comprising a step of reducingthe moisture content from liquid compositions of cyclophosphamide.

Further embodiment includes stable liquid formulations ofcyclophosphamide wherein the liquid formulations are prepared by aprocess comprising the steps of

-   -   a) preparing the bulk solution of cyclophosphamide by dissolving        cyclophosphamide in a suitable solvent.    -   b) incubating the bulk solution of cyclophosphamide solution of        step a) with adsorbents or mixture of adsorbents in suitable        ratio for suitable period of time.    -   c) filtering the cyclophosphamide solution from step b) by using        suitable filter followed by filling into vials, stoppering and        sealing.

In an embodiment the invention includes stable liquid formulations ofcyclophosphamide wherein the liquid formulations are prepared by aprocess comprising a step of reducing the moisture content from theliquid compositions of cyclophosphamide, wherein the process comprisingthe steps of

-   -   a) preparing the solution of cyclophosphamide by dissolving        cyclophosphamide in a suitable solvent.    -   b) preparing at least one column by using suitable adsorbent or        mixture of adsorbents. c) passing the cyclophosphamide solution        from step a) through the column or series of columns of step b).    -   c) optionally filtering the solution through a suitable filter        followed by filling into vials, stoppering and sealing.

In an embodiment the invention includes stable liquid formulations ofcyclophosphamide wherein the liquid formulations are prepared by aprocess wherein the cyclophosphamide solution is passed through thecolumn of adsorbents in a recirculation mode.

In an embodiment the invention includes stable liquid formulations ofcyclophosphamide wherein the liquid formulations are prepared by aprocess wherein the cyclophosphamide solution is passed through thecolumn of adsorbents at a flow rate ranging from about 0.1 mL/minute toabout 5 L/minute.

In yet another embodiment the invention includes stable liquidformulations of cyclophosphamide prepared by a process comprising a stepof reducing moisture content from both cyclophosphamide and liquidcompositions of cyclophosphamide.

In an embodiment the invention includes stable liquid formulations ofcyclophosphamide wherein the liquid formulations are prepared by aprocess comprising the steps of

-   -   a) reducing water content of cyclophosphamide by suitable drying        methods.    -   b) preparing the solution of cyclophosphamide by dissolving        cyclophosphamide of step a) in a suitable solvent.    -   c) preparing at least one column by using suitable adsorbent or        mixture of adsorbents.    -   d) passing the cyclophosphamide solution from step b) through        the column or series of columns of step c).    -   e) optionally filtering the solution through a suitable filter        followed by filling into vials, stoppering and sealing.

In one of the embodiment the invention includes stable liquidformulations of cyclophosphamide wherein the suitable solvent selectedfrom the group comprising of alcohol, polyethylene glycol, propyleneglycol, dimethyl acetamide, glycerol, polysorbate 80, polyethoxylatedcastor oil or combinations thereof.

In an embodiment suitable solvent is alcohol or its combinationsthereof.

In an embodiment the suitable solvent is ethanol or its combinations.

In a embodiment the invention includes stable liquid formulations ofcyclophosphamide wherein the moisture content from liquid compositionsof cyclophosphamide is reduced by means of adsorbents selected from thegroup comprising of molecular sieves, silica gel, activated alumina,activated charcoal or mixtures thereof.

In an embodiment the invention includes molecular sieves as adsorbent.

In an embodiment the invention includes the weight ratio ofcyclophosphamide bulk solution to adsorbent in the range of about 1:0.01to about 1:50.

In an embodiment the invention includes the weight ratio ofcyclophosphamide bulk solution to adsorbent in the range of about 1:0.05to about 1:25.

In a embodiment the invention relates to a stable liquid formulations ofcyclophosphamide bulk solution to adsorbent in the range of about 1:0.1to 1:10.

In an embodiment the invention includes liquid formulations ofcyclophosphamide, wherein moisture content of liquid formulation is lessthan about 5% by weight of the composition.

In an embodiment the invention includes liquid formulations ofcyclophosphamide, wherein moisture content of liquid formulation is lessthan about 2% by weight of the composition.

In an embodiment the invention includes liquid formulations ofcyclophosphamide, wherein moisture content of liquid formulation is lessthan about 2% by weight of the composition when stored at 2-8° C. for atleast 6M

In an embodiment the invention relates to stable liquid formulations ofcyclophosphamide wherein levels of impurity A in the formulation is lessthan about 1.5% by weight of label content of cyclophosphamide or itshydrate.

In an embodiment the invention relates to stable liquid formulations ofcyclophosphamide wherein levels of impurity B in the formulation is lessthan about 1.5% by weight of label content of cyclophosphamide or itshydrate.

In an embodiment the invention relates to stable liquid formulations ofcyclophosphamide wherein levels of impurity D in the formulation is lessthan about 1.5% by weight of label content of cyclophosphamide or itshydrate.

In an embodiment the invention relates to stable liquid formulations ofcyclophosphamide wherein levels of specified impurity at 0.21 RRT in theformulation is less than about 1.5% by weight of label content ofcyclophosphamide or its hydrate.

In an embodiment the invention relates to stable liquid formulations ofcyclophosphamide wherein levels of specified impurity at 0.55 RRT in theformulation is less than about 2.5% by weight of label content ofcyclophosphamide or its hydrate.

In an embodiment the invention relates to stable liquid formulations ofcyclophosphamide wherein levels of specified impurity at 0.75 RRT in theformulation is less than about 2.5% by weight of label content ofcyclophosphamide or its hydrate.

In one of the embodiment the invention relates to stable liquidformulations of cyclophosphamide wherein the total impurities are lessthan about 6% after storage at 2-8° C. for at least 6 months.

In one of the embodiment the invention includes stable liquidformulations of cyclophosphamide wherein the liquid formulations areprepared by a process comprising the steps of

a) preparing the bulk solution of cyclophosphamide by dissolvingcyclophosphamide in ethanol

b) incubating the bulk solution of cyclophosphamide of step a) withmolecular sieves until the moisture content of the solution is less thanabout 2.0% by weight of the composition.

c) filtering the cyclophosphamide solution from step b) by usingsuitable filter.

In an embodiment the invention includes stable liquid formulations ofcyclophosphamide wherein the liquid formulations are prepared by aprocess comprising the steps of

-   -   a) preparing the bulk solution of cyclophosphamide by dissolving        cyclophosphamide in ethanol.    -   b) preparing the column by using suitable molecular sieves.    -   c) passing the cyclophosphamide solution from step a) through        the column of step b) in a recirculation mode until moisture        content of the formulation is less than about 2.0% by weight of        the composition.    -   d) optionally filtering the solution through a suitable filter.

In one of the embodiment the invention includes stable liquidformulations of cyclophosphamide wherein the liquid formulation isintended for parenteral administration.

In an embodiment the invention includes stable liquid formulations ofcyclophosphamide wherein the liquid formulation is ready-to-useformulation.

In an embodiment the invention includes stable liquid formulations ofcyclophosphamide wherein the liquid formulation is ready-to-diluteformulation.

In an embodiment the invention relates to methods of using stable liquidformulations of cyclophosphamide in treating various cancers.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to stable liquid formulations ofcyclophosphamide and processes to prepare the stable liquid formulationsof cyclophosphamide. The invention relates to stable pharmaceuticalliquid formulations of cyclophosphamide comprising cyclophosphamide andat least one pharmaceutically acceptable excipient wherein moisturecontent of the liquid formulation is less than about 2.0% by weight. Theinvention also relates to stable liquid formulations of cyclophosphamideprepared by a process comprising a step of reducing the moisture contentfrom cyclophosphamide or liquid compositions of cyclophosphamide orboth. The invention further relate to method of using such stable liquidformulations of cyclophosphamide for treating various cancer disorders.

The various cancer disorders according to the invention includesMalignant lymphomas (Stages III and IV of the Ann Arbor staging system),Hodgkin's disease, lymphocytic lymphoma (nodular or diffuse), mixed-celltype lymphoma, histiocytic lymphoma, Burkitt's lymphoma; Multiplemyeloma; Leukemias: Chronic lymphocytic leukemia, chronic granulocyticleukemia (it is usually ineffective in acute blastic crisis), acutemyelogenous and monocytic leukemia, acute lymphoblastic (stem-cell)leukemia in children (cyclophosphamide given during remission iseffective in prolonging its duration); Mycosis fungoides (advanceddisease); Neuroblastoma (disseminated disease); Adenocarcinoma of theovary; Retinoblastoma; Carcinoma of the breast; biopsy proven “minimalchange” nephrotic syndrome in children.

The liquid formulations according to the invention possess number ofadvantages as compared to solutions prepared from sterile powders orlyophilizate immediately before use, they are a) less likely to becontaminated by particles or microbes b) render the dissolution stepsuperfluous and may be used immediately; c) contribute to the safety ofthe nursing staff who is handling the reconstitution or administrationof the drug and are more economical to prepare.

The term “cyclophosphamide” is intended to include any of thealternative forms in which cyclophosphamide can be administered such assalts, esters, anhydrous, hydrates such as monohydrate or dihydrate,solvates, crystalline or amorphous polymorphs, racemic mixtures,enantiomeric isomers and so on unless it is restricted to specificproperty for example cyclophosphamide with moisture content less than 5%by weight.

The terms ‘stable’ or ‘stability’ as used herein relate to both physicaland chemical stability, wherein cyclophosphamide can be stored forcommercially significant periods, such as at least 3 months, 6 months, 1year, or 2 years or 3 years, without significant physical instability(description, clarity etc) and chemical degradation. The stable mayrepresent stability when stored at 2-8° C. or at ambient conditions(e.g. 25° C.) or elevated temperatures (e.g. 40° C.). Percentdegradation may be determined by analyzing for impurities by suitableanalytical method.

The term “pharmaceutically acceptable” refers to ingredients that areuseful for preparing pharmaceutical compositions, and that is consideredto be generally safe, non-toxic and neither biologically nor otherwiseundesirable, and includes those ingredients acceptable for veterinaryuse as well as human pharmaceutical use.

The term “substantial” as used to describe percentage of hydrates orsolvates of cyclophosphamide in the formulation includes at least about90%, or at least about 95%, or at least about 99% anhydrous form.

The term “moisture content” or water content or bound water refer to thewater content of the drug or tis formulation and are synonymously usedin the present invention. The moisture content may be bound or inunbound form.

The term “ready to use” or “RTU” composition is a stable formulations ofcyclophosphamide which are ready for administration which may be oral orparenteral administration.

The term “ready-to-dilute” or RTD composition is a stable liquidformulations that are to be diluted with the suitable diluent furtherfor oral or parenteral administration. Suitable diluents may includesterile water for injection, 0.9% sodium chloride, 0.45% sodiumchloride, 5% dextrose or combinations thereof.

The term “sterile” composition is one in which essentially all forms ofmicrobial life has been destroyed by an appreciable amount to meet thesterilization criteria outlined in the United States Pharmacopoeia.

The term “incubation” denotes that cyclophosphamide solution is incontact with the adsorbent for certain period of time or adsorbents areimmersed in bulk solution of drug for certain period of time.

The term “adsorbent” includes any substance used to remove or reducemoisture or water content from any other substance or its compositions.Substance may be drug or excipient or mixtures thereof. Sometimesadsorbents also refer to dessicants.

The term “pharmaceutically effective concentration” refers to anyconcentration of the drug showing its therapeutic effect.

The terms “anhydrous alcohol” or dehydrated alcohol” or “absolutealcohol” are used synonymously.

The bulk solution of the drug as discussed in this application refers toany solution prepared by dissolving drug in a suitable solventoptionally with stirring.

The term “composition” in the present invention refers to combination ofdrug along with at least one pharmaceutically acceptable excipient andused in preparing pharmaceutical formulations with no specificlimitations. The liquid compositions refer to the compositions in theliquid form.

The term “formulation” refers to pharmaceutical dosage forms containingcompositions of cyclophosphamide. The pharmaceutical formulations of thepresent invention can be prepared as solutions or suspensions oremulsion or dispersions or elixirs and so on presented in glass ampoulesor glass vials or any suitable devices.

The formulations of the present invention are particularly suitable foruse in parenteral administration, but it will be understood that thesolutions may have alternative uses. For example, they may be used asintermediates in the preparation of other pharmaceutical dosage forms.Similarly, they may have other routes of administration including oralor intranasal or inhalation. Injectable formulations may take any routeincluding intramuscular, intravenous or subcutaneous or intrathecal,intraarterial and so on.

Injectable formulations are frequently formulated as aqueous solutions,in which water is the primary excipient. Injectable formulations can beprepared in conventional forms, either as liquid solutions orsuspensions, solid forms suitable for solubilization or suspension inliquid prior to injection, or as emulsions. Sterile injectableformulations can be prepared according to techniques known in the artusing suitable carriers, dispersing or wetting agents, and suspendingagents. The injectable formulations may be sterile injectable solutionsor suspensions in a nontoxic, parenteral acceptable diluent or solvent.Injectable formulations can be used as ready-to-use or ready-to-dilutecompositions.

Cyclophosphamide being nitrogen mustard exhibit poor stability inaqueous solutions due to rapid degradation. Mainly for this reasoncyclophosphamide has historically been compounded as a sterile drypowder mixture of cyclophosphamide monohydrate for reconstitution withwater for Injection or as the lyophilized solid with mannitol excipientfor reconstitution with water for Injection. It has been also observedthat the reconstituted solutions with water are to be administeredimmediately. Both procedures require costly, extensive processing inproduction and time-consuming hazardous handling in preparation orreconstitution. Additionally, both compositions lead to costly waste dueto very short shelf-life of the reconstituted solutions. Consequently,portions not used immediately must be discarded.

Due to inherent instability of cyclophosphamide in aqueous solutions,different non-aqueous solvents have been explored to prepare the liquidformulation. With the use of non-aqueous solvents, the stability ofcyclophosphamide was improved when compared to aqueous preparations butit was not promising on stability. Even in presence of non-aqueoussolvents (which contribute to very less quantity of moisture or watercontent) also cyclophosphamide was not that stable. It was understoodthat probably the bound water of cyclophosphamide monohydrate(approximately 6.25%) may be responsible for hydrolytic degradationcyclophosphamide in non-aqueous solvent such as anhydrous ethanol. Thereare different techniques to remove or reduce water from thecyclophosphamide such as lyophilization, vacuum drying, solventevaporation, adsorbents such as molecular sieve, silica gel, activatedalumina, activated charcoal etc.

In one of the embodiment the invention includes stable liquidformulations of cyclophosphamide comprising cyclophosphamide and atleast one pharmaceutically acceptable excipient.

In a embodiment the invention relates to stable liquid formulations ofcyclophosphamide comprising cyclophosphamide in pharmaceuticallyeffective concentration and at least one pharmaceutically acceptableexcipient.

In an embodiment the invention relates to stable liquid formulations ofcyclophosphamide wherein cyclophosphamide used in the formulation issubstantially in anhydrous form.

In an embodiment the invention includes the stable liquid formulationsof cyclophosphamide comprising cyclophosphamide with moisture contentless than about 5% by weight of the composition or is substantiallyanhydrous form.

In an embodiment the invention includes the stable liquid formulationsof cyclophosphamide comprising cyclophosphamide with moisture contentless than about 2% by weight of the composition.

In an embodiment the invention includes the stable liquid formulationsof cyclophosphamide comprising cyclophosphamide with moisture contentless than about 2% by weight of the composition when stored at 2-8° C.for at least 6M.

In an embodiment the invention covers stable liquid formulations ofcyclophosphamide wherein pharmaceutically effective concentration ofcyclophosphamide is at least 0.1 g per mL.

In an embodiment the invention covers stable liquid formulations ofcyclophosphamide wherein pharmaceutically effective concentration ofcyclophosphamide is at least 0.5 g per mL.

It has been observed that when the bound water was reduced fromcyclophosphamide monohydrate by subjecting to vacuum drying, thecyclophosphamide with reduced water content showed better stability thanits monohydrate form in anhydrous ethanol. This further confirms thatthe bound water of cyclophosphamide monohydrate may be responsible fordegradation.

In an embodiment the invention includes stable liquid formulations ofcyclophosphamide prepared by a process comprising a step of reducing themoisture content from cyclophosphamide.

In another embodiment the invention includes process for preparing theliquid formulations of cyclophosphamide wherein process comprising thesteps of

-   -   a) reducing water content of cyclophosphamide by suitable drying        methods.    -   b) preparing the solution of cyclophosphamide by dissolving        cyclophosphamide with reduced water content of step-a) in a        suitable solvent. Optionally the solution is filtered through a        suitable filter.    -   c) filling bulk solution of step-b) in vials followed by        sealing.

In an embodiment the invention includes stable liquid formulations ofcyclophosphamide wherein the moisture content of cyclophosphamide isreduced by selecting a suitable drying process selected from the groupcomprising of vacuum drying, lyophilization, solvent evaporation.

In one of the embodiment stable liquid formulations of cyclophosphamideprepared by removing the moisture content from cyclophosphamide byvacuum drying.

In another embodiment the invention includes stable liquid formulationsof cyclophosphamide prepared by a process comprising a step of reducingthe moisture content from liquid compositions of cyclophosphamide.

In another embodiment the invention includes stable liquid formulationsof cyclophosphamide prepared by a process comprising a step of reducingthe moisture content from both cyclophosphamide and the liquidcompositions of cyclophosphamide.

In an embodiment the invention includes stable liquid formulations ofcyclophosphamide wherein the moisture content from liquid compositionsof cyclophosphamide is reduced by means of adsorbents selected from thegroup comprising of molecular sieves, silica gel, activated alumina,activated charcoal.

In an embodiment the invention includes process of reducing the moisturecontent from liquid formulations of cyclophosphamide by using molecularsieves.

Molecular sieve are pelleted, beaded and powdered material, made fromthree dimensional materials. A molecular sieve is a material with verysmall holes of precise and uniform size. These holes are small enough toblock large molecules while allowing small molecules to pass. The smallmolecules are efficient to pass through the pores and when activatedthey becomes a powerful adsorbents in a wide range of operatingconditions with a strong absorption ability with water, hydrogen,oxygen, carbon dioxide and other polar molecules. The term “activated”with respect to adsorbents refer to the process wherein the molecularsieves are heated to certain temperature for certain period of time. Forexample molecular sieves when heated at 120° C. for about 12 hours maybe referred to as activated molecular sieves.

Molecular sieves are used as adsorbent for gases and liquids. Moleculessmall enough to pass through the pores are adsorbed while largermolecules are not. It is different from a common filter in that itoperates on a molecular level and traps the adsorbed substance. Forinstance, a water molecule may be small enough to pass through the poreswhile larger molecules are not, so water is forced into the pores whichact as a trap for the penetrating water molecules, which are retainedwithin the pores. Because of this, they often function as a desiccant. Amolecular sieve can adsorb water up to 22% of its own weight. Theprinciple of adsorption to molecular sieve particles is somewhat similarto that of size exclusion chromatography, except that without a changingsolution composition, the adsorbed product remains trapped because, inthe absence of other molecules able to penetrate the pore and fill thespace, a vacuum would be created by desorption.

There are different types of molecular sieves available. Molecularsieves can be microporous, mesoporous, or macroporous material.

-   -   Microporous (<2 nm) eg: Zeolites (aluminosilicate minerals;        Porous glass; Active carbon: Clays such as Montmorillonite,        Halloysite etc    -   Mesoporous material (2-50 nm) eg: Silicon dioxide (used to make        silica gel).    -   Macroporous material (>50 nm) eg: Mesoporous silica

Further there are different models available for molecular sieves basedon their adsorption capabilities including pore diameter, bulk density,adsorbed water etc. For example:

-   -   13× and 10× Molecular Sieves (Beads, Pellets and Powder)    -   3A Molecular Sieves (Beads, Pellets and Powder)    -   4A Molecular Sieves (Beads, Pellets and Powder)    -   5A Molecular Sieves (Beads, Pellets and Powder)

Among the available models of molecular sieves, type 3A molecular sievewas used for removing or reducing water from liquid formulationconsidering its unique pore size.

By using molecular sieves the water can be removed or reduced bydifferent methods such as

-   -   Static process wherein adsorbent or mixture of adsorbents in        certain weight ratio were incubated or immersed in the        cyclophosphamide bulk solution.    -   Dynamic process wherein the bulk solution of cyclophosphamide        was passed through the column or series of columns containing        adsorbent or mixture of adsorbents. The dynamic process can        further be in a recirculation method wherein the bulk solution        was continuously recirculated through the column using        peristaltic pump. Recirculation will increase the efficiency of        absorption.

In one of the embodiment the invention includes the stable liquidformulations of cyclophosphamide wherein weight ratio ofcyclophosphamide bulk solution to adsorbent is 1:0.01 to 1:50.

In an embodiment the invention includes the stable liquid formulationsof cyclophosphamide wherein weight ratio of cyclophosphamide bulksolution to adsorbent is 1:0.05 to 1:25.

In one of the embodiment the invention includes the stable liquidformulations of cyclophosphamide wherein weight ratio ofcyclophosphamide bulk solution to adsorbent is 1:0.1 to 1:10.

In one of the embodiment the invention includes the stable liquidformulations of cyclophosphamide wherein weight ratio ofcyclophosphamide bulk solution to adsorbent is 1:0.1 to 1:5.

In one of the embodiment the invention includes the stable liquidformulations of cyclophosphamide wherein weight ratio ofcyclophosphamide bulk solution to adsorbent is 1:0.1 to 1:2

In one of the embodiment the adsorbent is molecular sieves.

In another embodiment the invention includes the stable liquidformulations of cyclophosphamide, wherein moisture content of liquidformulation is less than about 5% by weight of the composition.

In another embodiment the invention includes the stable liquidformulations of cyclophosphamide, wherein moisture content of liquidformulation is less than about 2% by weight of the composition.

In another embodiment the invention includes the stable liquidformulations of cyclophosphamide, wherein moisture content of liquidformulation is less than about 1% by weight of the composition.

In one of the embodiment the invention includes the pharmaceuticalliquid formulations of cyclophosphamide comprising cyclophosphamide andat least one pharmaceutically acceptable excipient.

In an embodiment the invention includes stable liquid formulations ofcyclophosphamide comprising cyclophosphamide and at least onepharmaceutically acceptable excipient is non-aqueous solvent.

The various non-aqueous solvents suitable for the formulations of thepresent invention include but not limited to alkyl alcohols, forexample, ethanol/anhydrous ethanol/dehydrated alcohol/absolute alcohol,ethylene glycol, propylene glycol, butylene glycol, glycerin orglycerol, polysorbates, for example TWEEN 20, TWEEN 40, and TWEEN 80,and cyclodextrins (such as hydroxypropyl-.beta.-cyclodextrin),polyalkylene glycols, such as polyethylene glycol, polypropylene glycol,and polybutylene glycol, diemthyl acetamide, niacinamide, a diol such asa straight chain, branched or cyclic aliphatic diol, a triol such asstraight chain, branched or cyclic aliphatic triol, a polyoxyethyleneether and a polyethylene glycol ether.

The aliphatic diol may have either a straight chain or a branched chainand have 2-20 carbon atoms. Exemplary suitable aliphatic diols include1,2-propane diol, 1,3-propane diol, 1,4-butane diol, 1,5-pentane dioland its higher aliphatic homologs and their positional isomers. Thealiphatic cyclic diol may have from 5 to 20 carbon atoms. Exemplarysuitable aliphatic cyclic diols include 1,2-cyclopentane diol,1,2-cyclohexane diol and its higher aliphatic homologs and theirpositional isomers. The aliphatic triol may have from 3 to 20 carbonatoms and may have a straight chain or a branched chain Exemplarysuitable aliphatic triols include glycerin (glycerol) and its higheraliphatic homologs and their positional isomers like butane 1,2,3-triol,1,3,5-pentane triol. The aliphatic cyclic triol may have from 5 to 20carbon atoms. Exemplary aliphatic cyclic triols include cyclohexanetriol, cycloheptanetriol its higher aliphatic homologs and all theirpositional isomers. The polyoxy ethylene ether, exemplarypolyoxyethylene ethers include polysorbate-20 (Tween-20), polysorbate-40(Tween-40), polysorbate-60 (Tween-60), and polysorbate-80 (Tween-80).The polyethylene glycol ether, exemplary polyethylene glycol ethersinclude polyethoxylated castor oil, such as Cremophor® and other polyethers in that class.

In an embodiment the invention includes stable liquid formulations ofcyclophosphamide wherein excipient is at least one non-aqueous solventselected from the group comprising ethanol, propylene glycol,polyethylene glycol, dimethyl acetamide, glycerol, polysorbate 80,polyethoxylated castor oil or combinations thereof.

In an embodiment the invention relates to stable liquid formulations ofcyclophosphamide wherein non-aqueous solvent is ethanol.

In an embodiment the invention relates to stable liquid formulations ofcyclophosphamide wherein non-aqueous solvent is anhydrous ethanol.

In another embodiment the invention includes stable liquid formulationsof cyclophosphamide wherein ethanol is in the concentration of 10 to100% by weight of the composition.

In another embodiment the invention includes stable liquid formulationsof cyclophosphamide wherein non-aqueous solvent system is combination ofethanol such as glycerol with ethanol or propylene glycol with ethanolor polyethylene glycol with ethanol or polysorbate with ethanol orcremophor with ethanol.

In an embodiment the invention includes pharmaceutical stable liquidformulations of cyclophosphamide, wherein non aqueous solvent orcombination of non-aqueous solvents are present in the formulation in arange from about 10 to 100% by weight.

In one of the embodiment the invention includes stable liquidformulations of cyclophosphamide wherein the liquid formulations areprepared by a process comprising the steps of

-   -   a) preparing the bulk solution of cyclophosphamide by dissolving        cyclophosphamide in ethanol    -   b) incubating the bulk solution of cyclophosphamide of step a)        with molecular sieves until the moisture content of the solution        is less than about 2.0% by weight of the composition.    -   c) filtering the cyclophosphamide bulk solution from step b) by        using suitable filter followed by filling into vials, stoppering        and sealing.

In an embodiment the invention includes stable liquid formulations ofcyclophosphamide wherein the liquid formulations are prepared by aprocess comprising steps of

-   -   a) preparing the bulk solution of cyclophosphamide by dissolving        cyclophosphamide in ethanol.    -   b) preparing the column by using suitable grade of molecular        sieves.    -   c) passing the cyclophosphamide solution from step a) through        the column of step b) in a recirculation mode until moisture        content of the formulation is less than about 2.0% by weight of        the composition.    -   d) optionally filtering the solution through a suitable filter        then followed by filling into vials, stoppering and sealing.

In an embodiment the invention includes stable liquid formulations ofcyclophosphamide wherein the liquid formulations are prepared by aprocess comprising the steps of

-   -   a) reducing water content of cyclophosphamide by suitable drying        methods.    -   b) preparing the bulk solution of cyclophosphamide by dissolving        cyclophosphamide of step a) in a suitable solvent.    -   c) preparing at least one column by using suitable adsorbent or        mixture of adsorbents. d) passing the cyclophosphamide solution        from step b) through the column or series of columns of step c).    -   d) optionally filtering the solution through a suitable filter        followed by filling into vials, stoppering and sealing.

In an embodiment the invention includes stable liquid formulations ofcyclophosphamide, wherein the cyclophosphamide solution is prepared bydissolving cyclophosphamide in suitable solvent by stirring at 50 to2000 rpm speed.

In one of the embodiment the invention includes stable liquidformulations of cyclophosphamide wherein the liquid formulations areprepared by a process which is under continuous nitrogen purging.

In an embodiment the invention includes stable liquid formulations ofcyclophosphamide prepared by dissolving cyclophosphamide in a suitablesolvent purged with nitrogen followed by filling into glass vial,stoppered and sealing.

In one of the embodiment the invention includes stable liquidformulations of cyclophosphamide wherein the liquid formulation isintended for parenteral administration.

In an embodiment the invention includes stable liquid formulations ofcyclophosphamide wherein the liquid formulation is ready-to-useformulation.

In an embodiment the invention includes stable liquid formulations ofcyclophosphamide wherein the liquid formulation is ready-to-diluteformulation.

From the degradation data it has been observed that the degradants areformed by attack on the carbon containing chlorine atom. By includingchloride ion source in the formulation the formation of degradants byattack on the carbon atom can be minimized. Suitable chloride ion sourceincludes sodium chloride, potassium chloride, hydrochloric acid or anyother source of chloride ion that is sufficiently soluble in the chosenformulation solvent. In an embodiment the chloride ion source is presentin the range of about 0.01% to about 15% w/w of the formulation.

However chloride ion sources for example sodium chloride or potassiumchloride etc. is soluble in water and has limited solubility innon-aqueous solvents. To stabilize cyclophosphamide chloride ion sourceshould be in solubilized form. Hence while preparing the stable liquidformulation of cyclophosphamide with chloride ion source, identificationof suitable non aqueous solvent wherein chloride ion is in solubilizedform to stabilize the cyclophosphamide is critical and important.

In an embodiment the invention includes the process of preparing theliquid formulations wherein process comprising the steps of

-   -   a) preparing the bulk solution of cyclophosphamide by dissolving        cyclophosphamide in nitrogen purged first non-aqueous solvent.    -   b) dissolving chloride ion source in nitrogen purged second        non-aqueous solvent.    -   c) mixing step a) and step b)    -   d) filling the bulk solution into glass vials followed by        stoppering and sealing.

In one of the embodiment the invention relates to stable ready to useinjectable liquid formulations of cyclophosphamide, wherein formulationscomprise cyclophosphamide and non-aqueous solvent or solvent system suchthat the chloride ion source is in solubilized form.

In an embodiment the invention includes the stable ready to useinjectable liquid formulations wherein formulation comprise non-aqueoussolvent or solvent system that is capable of keeping chloride ion sourcein solubilized form is glycerol or propylene glycol or combinationthereof.

The pH of the non-aqueous liquid plays a crucial role in the stabilityof the nitrogen mustard formulation. Protonation of the nitrogen in themustard moiety avoids the formation of an aziridine ring, which ishighly unstable and can result in unacceptable levels of degradation ofthe nitrogen mustard. An acidic pH is required to maintain theprotonated state of the nitrogen in the mustard moiety. In anembodiment, the pH of the formulation is in a range between about pH 3to about pH 9.

In an embodiment the invention includes the liquid formulations ofcyclophosphamide with at least one pH adjusting agents or at least onebuffering agent

The pH-adjusting agents may include pharmaceutically acceptable acids,bases, or buffering agents. For example, the acids may include, but arenot limited to, one or more inorganic mineral acids such ashydrochloric, hydrobromic, sulfuric, phosphoric, nitric, and the like;or one or more organic acids such as acetic, succinic, tartaric,ascorbic, citric, fumaric, oxalic, maleic acie, adipic acid, glutamic,benzoic, methanesulphonic, ethanesulfonic, trifluoroacetic, hydroxy acidor alpha hydroxy acids and the like. The bases may be one or moreinorganic bases or organic bases, including, but not limited to,alkaline carbonate, alkaline bicarbonate, alkaline earth metalcarbonate, alkaline hydroxide, alkaline earth metal hydroxide or aminesuch as ethanolamine. For example, the inorganic or organic base may bean alkaline hydroxide such as lithium hydroxide, potassium hydroxide,cesium hydroxide, sodium hydroxide or the like; an alkaline carbonatesuch as calcium carbonate, sodium carbonate or the like; or an alkalinebicarbonate such as sodium bicarbonate or the like; the organic base mayalso be sodium acetate

Buffering agents may comprise pharmaceutically acceptable reagents orcomponents that contribute to maintaining pH between 3 to 9. Suitablebuffering agents include but not limited to ascorbate, lactobionate,gentisate, succinate, .alpha.-lipoic acid, maleate, chloroacetate,citrate, bicarbonate, tartrate, glycylglycine, formate, benzoate,phosphate, citrate, lactate, acetate, propionate, pyridine, piperazine,pyrophosphate, histidine, 2-(N-morpholino)ethanesulfonic acid (“MES”),cacodylic acid, (bis(2-hydroxyethyl)-imino-tris(hydroxymethyl)-methane)(“bis-TRIS”), bicarbonate, or a combination of these buffering agents

Additional excipients that can be included in the liquid formulations ofthe present invention include antioxidants, preservatives, polymers,sugars or polyols or combination thereof. Suitable antioxidants includebut not limited to butylated hydroxytoluene, butylated hydroxy anisole,alpha.-tocopherol, citric acid, ascorbic acid, monothiogleycerol, sodiumsulfite, sodium metabisulfite, thymol, propyl gallate, histidine,methionine and combinations thereof. The antioxidant may be present at arange of about 0.01% w/w to about 10% w/w of the formulation.

Suitable buffering agent include but not limited to acetate, tartrate,ascorbate, lactobionate, gentisate, succinate, lactate, α-lipoic acid orany combinations thereof. Suitable polymers include poloxamers,hydroxyethyl starch, polyvinyl pyrrolidone or combination thereof.Suitable preservatives include but not limited to methyl-, ethyl- andpropyl parabens or any combination thereof. Suitable polyols include butnot limited to sucrose, dextrose, dextrin, propylene glycol, sorbitol,glycerol or any combinations thereof.

In various embodiments the composition may further include one of moretonicity modifying agents such as sodium chloride, dextrose, boric acid,sodium tartrate, propylene glycol, polyols (such as mannitol andsorbitol), or other inorganic or organic solutes. inorganic salts,organic salts or combination thereof. Apart from sodium chloride, theother inorganic salts may comprise potassium chloride, magnesiumchloride, calcium chloride and the organic salts may comprise conjugatebase of trifluoroacetic acid.

As discussed above, cyclophosphamide is highly unstable in presence ofmoisture/water content and/or temperature. From the forced degradationdata of cyclophosphamide, it has been observed that six known impuritiesnamely Impurity A, B, D and three unknown specified impurities areobtained under different stressed conditions as shown below

-   -   a) Impurity A is formed in acidic conditions. Impurity A,        chemically Bis (2-Chloroethyl) amine hydrochloride and        represented by structure II.    -   b) Impurity B is formed due to basic hydrolysis. Impurity B,        chemically        3-(2-Chloroethyl)-2-oxo2-hydroxy-1,3,6,2-oxadiazaphosphone and        represented by structure III.    -   c) Impurity D chemically 3-[2-(2-chloroethylamino) ethylamino]        propyldihydrogen phosphate dihydrochloride and represented by        structure IV.

-   -   d) Specified impurity at 0.21 RRT.    -   e) Specified impurity at 0.55 RRT.    -   f) Specified impurity at 0.75 RRT.

In an embodiment the invention relates to stable liquid formulations ofcyclophosphamide wherein levels of impurity A in the formulation is lessthan about 1.5% by weight of label content of cyclophosphamide or itshydrate.

In an embodiment the invention relates to stable liquid formulations ofcyclophosphamide wherein levels of impurity B in the formulation is lessthan about 1.5% by weight of label content of cyclophosphamide or itshydrate.

In an embodiment the invention relates to stable liquid formulations ofcyclophosphamide wherein levels of impurity D in the formulation is lessthan about 1.5% by weight of label content of cyclophosphamide or itshydrate.

In an embodiment the invention relates to stable liquid formulations ofcyclophosphamide wherein levels of specified impurity at 0.21 RRT in theformulation is less than about 1.5% by weight of label content ofcyclophosphamide or its hydrate.

In an embodiment the invention relates to stable liquid formulations ofcyclophosphamide wherein levels of specified impurity at 0.55 RRT in theformulation is less than about 2.5% by weight of label content ofcyclophosphamide or its hydrate.

In an embodiment the invention relates to stable liquid formulations ofcyclophosphamide wherein levels of specified impurity at 0.75 RRT in theformulation is less than about 2.5% by weight of label content ofcyclophosphamide or its hydrate.

In an embodiment the invention relates to stable liquid formulations ofcyclophosphamide wherein total drug related impurities in theformulation is less than 6% by weight of label content ofcyclophosphamide or its hydrate.

In one of the embodiment the invention relates to stable liquidformulations of cyclophosphamide wherein the total impurities are lessthan about 6% after storage at 2-8° C. for at least 3 months or at least6 months.

The moisture content from the cyclophosphamide can be removed byapplying any suitable drying technique such as lyophilization, vacuumdrying, solvent evaporation, use of adsorbents such as molecular sieves,activated charcoal, activated alumina, silica gel and so on.

In an embodiment the invention includes pharmaceutical liquidformulations of cyclophosphamide, wherein cyclophosphamide is at thepharmaceutically effective concentration in the range of about 0.1 mg/mLto about 5 g/mL.

In an embodiment the invention includes pharmaceutical liquidformulations of cyclophosphamide, wherein cyclophosphamide is at thepharmaceutically effective concentration of at least 0.1 g/ml.

In an embodiment the invention includes pharmaceutical liquidformulations of cyclophosphamide, wherein cyclophosphamide is at thepharmaceutically effective concentration of at least 0.5 g/ml.

To prepare the pharmaceutical dosage form, the pharmaceuticalformulation can be packaged in the container by any suitable methodknown in the art.

The solutions are often required to filter to remove the unwantedparticles from it. For this a suitable filters such as PVDF filters ofsize 0.2 micron may be used.

In embodiments, the invention provides methods of filling containersthat contain a solution of cyclophosphamide or salts or hydrates,comprising: a) providing one or more open containers; b) filing thecontainers with a solution of cyclophosphamide optionally in an asepticenvironment; c) sealing the filled containers; and d) sterilizing thesealed, filled containers.

The liquid compositions of cyclophosphamide can be contained within asealed container. More preferably, the container is provided with anopening and means for aseptically sealing the opening, e.g, such thatthe sealed container is fluidly sealed or the sealed opening issubstantially impermeable to atmospheric gases, moisture, pathogenicmicroorganisms or the like. The container can be constructed anysuitable material such as, for example glass, polypropylene,polyethylene terephthalate, and the like. In a preferred embodiment thecontainer is constructed of glass. Suitable glass vials include moldedand tubing glass vials such as, for example, Type I molded glass vials,and the like.

A suitable means for sealing the container can include, for example, astopper, a cap, a lid, a closure, a covering which fluidly seals thecontainer, or the like. The means for sealing the container are notlimited to separate closures or closure devices. In a embodiment, themeans for aseptically sealing the container includes a stopper such as,for example, a stopper that is configured to fluidly seal the opening.Suitable stoppers include conventional medical grade stoppers which donot degrade or release significant amounts of impurities. Some of thestopper materials include silicone rubber, Teflon coated stoppers,slotted bromobutyl rubber, etc.

Optionally, an outer seal is provided which covers and entirelysurrounds the stopper. The outer seal can be constructed of any suitablematerial. When an outer seal is used, it is preferably fitted with a lidthat can be easily manually removed to provide access to the stopper.Suitable outer seals can include, for example, Flip-offAluminum/Polypropylene Seals. Such seals include an outer rim made of asuitable material, such as aluminum, that entirely surrounds the lateraledge of the stopper and further include a lid (typically polypropyleneor other suitable material) that entirely covers the upper surface ofthe stopper. The polypropylene lid can be “flipped” off e.g., byexerting upward pressure with a finger or thumb, to provide access tothe stopper, e.g., so that it can be punctured with a hypodermic needleto withdraw the composition from the vial.

The other suitable devices for liquid formulations include but notlimited to, pre-filled syringes or pen devices or auto-injectors and soon.

The invention includes use of packaging materials such as containers andclosures of high-density polyethylene (HDPE), low-density polyethylene(LDPE) and or polypropylene and/or glass, glassine foil, polyvinylchloride, polyvinylidene dichloride, etc.

In yet another embodiment the invention includes the methods of usingthe liquid formulations of cyclophosphamide in treating cancers. Variouscancers include malignant lymphomas: Hodgkin's disease, lymphocyticlymphoma, mixed-cell type lymphoma, histiocytic lymphoma, Burkitt'slymphoma; multiple myeloma, leukemias, mycosis fungoides, neuroblastoma,adenocarcinoma of ovary, retinoblastoma, breast carcinoma. Theformulations of the present invention may also be extended to treatnephrotic Syndrome in Pediatric Patients who failed to adequatelyrespond to or are unable to tolerate adrenocorticosteroid Therapy.

Analytical Method:

-   -   1. Water content: Water content was estimated by karl fischer        (KF) method wherein suitable quantity of methanol was taken in        the flask and titrated with karl fischer reagent to neutralize        the methanol. Then weighed quantity of the sample is added to        the flask and titrated to the end point.    -   2. Impurities: The known impurities such as impurity A,B,D,        unknown specified impurities, unknown impurities and the total        impurities were estimated by the following analytical method:    -   Buffer: Potassium dihydrogen phosphate was taken in water and        the pH adjusted to pH 7.0 with diluted sodium hydroxide solution        followed by filtration through 0.45 μm filter.    -   Mobile Phase A: The buffer was used as mobile phase A.    -   Mobile phase B: The buffer and acetonitrile was mixed in 20:80        v/v ratios respectively.

Chromatographic Conditions:

-   -   Column: Waters symmetry shield RP-18 250×4.6 mm, 5 μm    -   Flow rate: 0.5 mL/min    -   Wavelength of detection: 195 nm    -   Column temperature: 25° C.±5° C.    -   Injection volume: 20 μL    -   Run time: 80 minutes

Diluent: HPLC Grade Water.

Gradient Program:

Time (Minutes) Mobile Phase-A % Mobile Phase-B % 0 100 0 10 100 0 40 5050 55 20 80 65 20 80 70 100 0 80 100 0

Representative relative retention factor (RRF) and relative retentiontime (RRT) are represented as below:

Name of the compound RRT RRF Cyclophosphamide 1.00 1.00 Impurity A 0.911.10 Impurity B 0.15 0.82 Impurity D 0.18 0.44 Specified impurity at0.21 0.21 — Specified impurity at 0.55 0.55 — Specified impurity at 0.750.75 —

The following examples further describe certain specific aspects andembodiments of the invention and demonstrate the practice and advantagesthereof. It is to be understood that the examples are provided only forpurposes of illustration and are not intended to limit the scope of theinvention in any manner.

EXAMPLES Comparative Example 1: Pharmaceutical Formulation ofCyclophosphamide Monohydrate [without Reducing the Moisture Content]

Composition:

Ingredients Quantity Cyclophosphamide monohydrate 500 mg Anhydrousethanol or dehydrated qs to 1 mL alcoholManufacturing Process:

-   1) Cyclophosphamide monohydrate was taken in vessel. 60% of    dehydrated alcohol was added to vessel with continuous stirring at    300-400 rpm until clear solution was obtained.-   2) Then remaining quantity of dehydrated alcohol was added to make    up the volume 100% and stirred for about 10 minutes to obtain a    clear homogeneous solution.-   3) Bulk solution was filtered through 0.2μ filter and filtered    solution was filled in 2 mL glass vial, stoppered and sealed.-   4) Sealed vials were charged on stability at 2-8° C. for about 6M    and 25° C./60% RH for about 2M. Table 1 show that stability data    generated.

TABLE 1 25° C./60% 2-8° Parameter Initial RH - 2M C. - 6M Moisturecontent (%) 4.05 3.74 4.0 Impurity B (%) 0.05 0.39 0.32 Impurity D (%) —0.94 0.37 Specified impurity at 0.21 RRT (%) ND 0.54 0.02 Specifiedimpurity at 0.55 RRT (%) 0.02 1.61 0.42 Specified impurity at 0.75 RRT(%) ND 1.08 0.25 Total impurities (%) 0.09 6.45 1.67

Example 1: Pharmaceutical Formulation of Cyclophosphamide with ReducedWater Content by Vacuum Drying

Ingredients Quantity Cyclophosphamide monohydrate* 500 mg Anhydrousethanol Qs to 1 mL *Water content of the input cyclophosphamidemonohydrate is 6.25% w/w.Manufacturing process:

-   1. The water content of cyclophosphamide monohydrate was reduced by    using vacuum drying. Cyclophosphamide monohydrate was filled in 50    ml glass vial and half stoppered then the weight of the vial was    taken and loaded into lyophilizer and subjected for drying under    vacuum condition in lyophilizer to reduce the water content of    monohydrate. Vials were checked for the final weight. Water content    in the vials was around approximately 1%.-   2. Anhydrous ethanol was taken and purged with nitrogen gas so that    dissolved oxygen is: 1 ppm.-   3. 70% of nitrogen purged anhydrous ethanol was taken.-   4. Cyclophosphamide monohydrate with reduced water content from    step 1) was added to ethanol of step-3 and dissolved by stirring at    1000 rpm for about 15 minutes or until clear solution forms under    continuous nitrogen purging.-   5. The volume was made up to 100% using nitrogen purged anhydrous    ethanol and stirred for about 10 minutes for uniform distribution.-   6. The bulk solution was filled in 2 ml glass vial and blanketed    with nitrogen in head space and stoppered with 13 mm stopper and    sealed with 13 mm seal.    Stability Data:    The sealed vials were exposed to 40° C., 25° C. and tested for    description (physical stability) and total impurities (chemical    stability) at 25° C. for 1 week, 2 week, 1 month, 3 months and at    40° C. for 1 week, 2 weeks. Table 2 shows the results.

TABLE 2 Stability condition Time period Description Total impurities (%)Initial Clear solution 0.06 25° C. 1 week Clear solution 0.20 2 weekClear solution 0.54 1 month Clear solution 0.97 3 months Clear solution2.8 40° C. 1 week Clear solution 0.84 2 week Clear solution 3.82

Example 2: Pharmaceutical Formulation of Cyclophosphamide with ReducedWater Content by Vacuum Drying

Composition: Same as that of Example 1

Manufacturing Process:

-   -   1) Cyclophosphamide monohydrate was filled in glass vial, half        stoppered and subjected to vacuum drying (in lyophilizer) at        25° C. at 50 mtorr pressure for about 4 hours to reduce the        moisture content of cyclophosphamide monohydrate.    -   2) This cyclophosphamide was added to 70% of dehydrated alcohol        and dissolved by stirring at 1000 rpm until clear solution        formed. Then the volume was made to 100% with the remaining        dehydrated alcohol.    -   3) Bulk solution was filled in 2 mL glass vial, stoppered and        sealed.    -   4) Sealed vials were charged for stability at 25° C. and 2-8° C.        for about 3 M.        Table 3 shows the stability data generated.

TABLE 3 25° C./60% 2-8° C. - Parameter Initial RH - 2M 3M Moisturecontent (%) 1.0  NA* NA Impurity B (%) 0.03 0.28 0.15 Impurity D (%) ND** 0.1  0.02 Specified impurity at 0.21 RRT (%) ND 0.11 ND Specifiedimpurity at 0.55 RRT (%) ND 0.54 0.16 Specified impurity at 0.75 RRT (%)ND 0.66 0.11 Total impurities (%) 0.06 1.96 0.49 *NA: Not available**ND: Not detectable

Example 3: Pharmaceutical Formulation of Cyclophosphamide with ReducedWater Content by Vacuum Drying

Composition and process same as that of example 1.

The sealed vials were charged for stability at 2-8° C. and 25° C./60% RHfor about 3 M.

Stability Data:

The samples exposed at 2-8° C. and 25° C. were tested for chemicalstability i.e. total impurities at 1W, 2W, 1M, 2M and 3M time intervalas shown in the below table 4.

TABLE 4 Stability condition Time period Total impurities (%) Initial0.05 2-8° C. 1 week 0.08 2 weeks 0.08 1 month 0.21 2 months 0.19 3months 0.49 25° C./60% RH 1 week 0.27 2 weeks 0.51 1 month 1.52 2 months1.96 3 months 3.92

Example 4: Pharmaceutical Formulation of Cyclophosphamide Prepared byStatic Process with the Ratio of Cyclophosphamide Bulk Solution toMolecular Sieves as 1:0.5

Ingredients Quantity Cyclophosphamide monohydrate 500 mg Anhydrousethanol q.s. to 1 mL Molecular Sieves Qs* *Quantity Sufficient.Manufacturing Process:

-   -   1. Anhydrous ethanol was added to cyclophosphamide monohydrate        and dissolved using magnetic stirrer.    -   2. After complete solubilization, the volume of solution was        measured and made up to desired volume using anhydrous ethanol        and the bulk solution was stirred for about 5 minutes.    -   3. Molecular sieves are added to the bulk solution of step 2)        and incubated at 2-8° C. for about 8 hours [weight ratio of        cyclophosphamide bulk solution to molecular sieves is 1:0.5 i.e.        1 part of cyclophosphamide bulk solution and 0.5 parts of        molecular sieves].    -   4. The bulk solution after 8 hours incubation was filtered        through 0.2 filter and analyzed for water content, assay and        total impurities at intermittent intervals of 2 hours, 4 hours,        6 hours, 8 hours. Results are captured in table 5

TABLE 5 Time interval Water content (%) Assay (%) Total impurities (%)Initial 3.54 102.3 0.07 2 hours 1.60 103.3 0.03 4 hours 0.87 102.9 0.036 hours 0.44 103.7 0.05 8 hours 0.24 104.3 0.04

Example 5: Pharmaceutical Formulation of Cyclophosphamide Prepared byStatic Process with Weight Ratio of Cyclophosphamide Bulk Solution toMolecular Sieves in 1:1 Ratio

Composition same as example 3.

Manufacturing Process:

-   -   1. Cyclophosphamide monohydrate was dissolved in anhydrous        ethanol using magnetic stirrer.    -   2. After complete solubilization, the volume of solution was        measured and made up to desired volume using anhydrous ethanol.        Stirring continued for about 5 minutes.    -   3. Bulk solution of step 2) and molecular sieves were taken in        the weight ratio of 1:1 i.e. 1 part of cyclophosphamide bulk        solution and 1 part of activated molecular sieve [Molecular        sieves heated at 120° C. for about 12 hours].    -   4. The bulk solution with molecular sieves incubated or immersed        in, was stored at 2-8° C. for about 8 hours.    -   5. After 8 hours the solution was filtered through 0.2μ filter        and filled in 2 mL glass and charged for stability at 2-8° C.        and 25° C./60% RH and analyzed for water content and total        impurities at a time points of 1M, 2M, 3M, 6M and the results        are captured in table 6.

TABLE 6 Time interval Water content (%) Total impurities (%) Initial0.57 0.25 2-8° C. 1 M  NA* 0.25 2-8° C. 2 M NA 0.32 2-8° C. 3 M NA 0.412-8° C. 6 M 0.64 1.15 25° C./60% RH 1 M NA 1.63 25° C./60% RH 2 M 0.532.89 25° C./60% RH 3 M NA 5.33 25° C./60% RH 6 M NA 8.82 *Not Analyzed.

Example 6: Pharmaceutical Formulation of Cyclophosphamide Prepared byStatic Process

Composition: Same as that of Example 1

Manufacturing Process:

-   -   1) 60% dehydrated alcohol was added to cyclophosphamide        monohydrate in a vessel with continuous stirring at 300-400 rpm        until clear solution was obtained. Then the volume was made to        100% with the remaining dehydrated alcohol.    -   2) Molecular sieves (ratio of cyclophosphamide bulk solution to        molecular sieve 1:0.5) were added to bulk solution.    -   3) After holding sample with molecular sieves for 8 hours, the        bulk solution was filtered. The filtered solution was filled in        glass vial, stoppered and sealed.    -   4) Sealed vials were charged on stability at 2-8° C. and 25°        C./60% RH. Table 7 shows that stability data of the formulation        in comparison with initial condition.

TABLE 7 25° C./60% 2-8° C. - Parameter Initial RH - 2M 6M Moisturecontent (%) 0.57  0.565  0.596 Impurity B (%) 0.11 0.30 0.22 Impurity D(%) ND 0.05 0.02 Specified impurity at 0.21 RRT (%) ND 0.25 ND Specifiedimpurity at 0.55 RRT (%) 0.05 0.74 0.34 Specified impurity at 0.75 RRT(%) 0.08 0.04 NA Total impurities (%) 0.09 2.86 1.26

Example 7: Pharmaceutical Formulation of Cyclophosphamide with VacuumDrying (Example 7A) and without Vacuum Drying (Example 7B) Prepared byStatic Process Using Molecular Sieves

Composition is same as that of example 4.

Manufacturing Process:

-   -   1) For example 7A, cyclophosphamide monohydrate was placed in        specially designed perforated trays which were kept at        lyophilizer and vacuum drying was carried out as following        conditions.

Step Shelf temp (° C.) Chamber pressure Time (minutes) Freezing Rate 22—  15 Hold 22 —  15 Phase-Primary drying Rate 22 50 120 Hold 22 50 960Total time 18 hrs. 30 minutes

-   -   2) Cyclophosphamide monohydrate with reduced water content of        step 1) (Example 5A) and without vacuum drying (Example 7B) was        dissolved in half the quantity of anhydrous ethanol with        stirring.    -   3) After complete solubilization remaining quantity of anhydrous        ethanol was added and made up the volume.    -   4) The bulk solution of step 3 was tested for water content and        was filled into vials. Half of the vials were incubated with        activated (molecular sieves heated at 120° C. for 12 hours)        molecular sieve and remaining half were incubated with        non-activated molecular sieves.    -   5) The bulk solution of step 4) is filtered through 0.2μ filter        and filled into glass vials.    -   6) All the vials were charged for stability at 2-8° C. & 25° C.        for about 2 & 5 hours respectively and tested for water content.        The water content data is being tabulated in table 8.

TABLE 8 Example 7A Example 7B Activated Non-activated ActivatedNon-activated Time 25° C. 2-8° C. 25° C. 2-8° C. 25° C. 2-8° C. 25° C.2-8° C. Initial 1.82 3.53 2 hour 0.43 0.54 0.35 0.22 1.10 0.84 0.87 0.825 hour 0.20 0.28 0.11 0.21 0.74 0.62 0.52 0.40

From the above data, it has been observed that the water content wassignificantly reduced from initial. Further the water content of theexample 5A prepared by using cyclophosphamide monohydrate with reducedwater content by vacuum drying along with molecular sieves in staticprocess showed more reduction in water content.

Example 8: Pharmaceutical Formulation of Cyclophosphamide Prepared byDynamic Process Using Molecular Sieves

Composition is same as that of example 4.

Manufacturing Process:

-   1) Cyclophosphamide was dissolved in 90% of anhydrous ethanol with    magnetic stirrer and volume was made up using remaining 10%    anhydrous ethanol.-   2) Molecular sieves were added in SS (stainless steel) column and    the anhydrous alcohol was passed through the column at flow rate of    10 mL/minute flow rate using peristaltic pump.-   3) The column was drained using peristaltic pump in reverse    direction and the nitrogen gas was drained through the column.-   4) The bulk solution of step 1) was passed through the column and    the samples were analyzed for water content after 15 minutes, 60    minutes, 120 minutes and 180 minutes. The water content data is    shown in below table 9.

TABLE 9 Time period Water content (%) Initial 3.8128  15 minutes 1.2871 60 minutes 0.3505 120 minutes 0.1674 180 minutes 0.1190 Final Bulksolution 0.2241

Example 9: Pharmaceutical Formulation of Cyclophosphamide Prepared byUsing Dynamic Process in Recirculation Method

Composition is same as that of example 4

Manufacturing Process:

-   -   1. Cyclophosphamide monohydrate was taken in a vessel.    -   2. Anhydrous ethanol was added to step-1 and cyclophosphamide        was dissolved by using magnetic stirrer.    -   3. After complete solubilization, the volume of solution was        made up by using anhydrous ethanol (2% excess anhydrous ethanol        was added in to compensate volume loss due to water removal).    -   4. Cyclophosphamide bulk solution and molecular sieves are taken        in 2:1 ratio. The molecular sieves type 3A was filled in SS        column (20 mm ID; 250 mm length).    -   5. The column is rinsed with anhydrous ethanol at 10 mL/min flow        rate using peristaltic pump.    -   6. The column is being drained by pumping liquid out of the        column at 10 mL/min.    -   7. Cyclophosphamide bulk solution of step 3) was pumped through        column of step 6) with flow rate 10 mL/min and the effluent was        recirculated to the feed container containing bulk solution of        cyclophosphamide.    -   8. The sampling is done of the bulk solution at different time        intervals for water content for a total of 150 minutes.    -   9. The water content of the formulation collected at different        time points have been analyzed and results are shown in table        10.

TABLE 10 Time interval Water content (%) Initial (Untreated) 3.5017  10min (effluent) 1.5674  10 min (Treated Bulk*) 2.2465  30 min (TreatedBulk) 1.2345  50 min (Treated Bulk) 0.8016  70 min (Treated Bulk) 0.5913 90 min (Treated Bulk) 0.4306 110 min (Treated Bulk) 0.3258 130 min(Treated Bulk) 0.2512 150 min (Treated Bulk) 0.2326 Final Treated Bulk**0.3492 *Treated bulk means bulk solution after 10 minutes ofrecirculation. **Final treated bulk is after draining out all thesolution from column & tubing's

Example 10: Pharmaceutical Formulation of Cyclophosphamide by ReducingMC Using Molecular Sieves (MS) Using Dynamic Process

Composition: Same as that of Example 4.

Manufacturing Process:

-   1) Molecular sieves [MS] column was prepared transferring molecular    sieves to dried stainless steel [SS] column.-   2) 60% of dehydrated alcohol was added to cyclophosphamide    monohydrate in a vessel with continuous stirring at 300-400 rpm    until clear solution was obtained. The volume was made up to 100%    using remaining dehydrated alcohol and stirred for about NLT 10    minutes to ensure complete mixing of the solution.-   3) The bulk solution was circulated through SS column in a    recirculation method until moisture content is below 0.5% w/w.-   4) Bulk solution was filtered through 0.22μ filter, filled,    stoppered and sealed. Sealed vials are subjected to stability at    2-8° C. and 25° C./60% RH. Table 11 shows the stability data.

TABLE 11 25° C./60% 2-8° C. - Parameter Initial RH - 2M 6M Moisturecontent (%) 0.2  0.2 0.2 Impurity B (%) 0.03 0.3 0.2 Impurity D (%) NDND ND Specified impurity at 0.21 RRT (%) ND 0.2  0.03 Specified impurityat 0.55 RRT (%) ND 0.1  0.07 Specified impurity at 0.75 RRT (%) NA NA NATotal impurities (%) 0.03 2.5 1.0

Example 11: Pharmaceutical Formulation of Cyclophosphamide Prepared byUsing Series of Columns

Composition is same as that of example 4.

Manufacturing Process:

-   1) Anhydrous ethanol was added to cyclophosphamide taken in a    vessel.-   2) The mixture was dissolved using magnetic stirrer. After complete    solubilization, the volume of solution was made up using anhydrous    ethanol.-   3) The molecular sieves are filled in two SS columns wherein both    the columns are connected in series.-   4) A portion of anhydrous ethanol was added to fill both the columns    followed by pumping a small portion of anhydrous ethanol through the    column at 6 mL/minute using peristaltic pump.-   5) The column is being drained by pumping liquid out of the column    at 6 mL/minute.-   6) The cyclophosphamide solution of the step 2) was pumped through    the column with flow rate 6 mL/minute and water content is tested    for each sample collected at different time points as shown below    table 12

TABLE 12 Time period Water content (%) Initial 3.5252 20 minutes 0.304435 minutes 0.4287 50 minutes 0.4426 65 minutes 0.4261 80 minutes 0.4322

Example 12-13: Pharmaceutical Liquid Formulation of Cyclophosphamide

Example-13 Example-14 Ingredients Quantity/mL Cyclophosphamide 500 mg500 mg Glycerol 0.2 mL 0.2 ml Sodium chloride — 2 mg Absolute Ethanol qsto 1 mL qs to 1 mLManufacturing Process:

-   -   1. Absolute Ethanol was purged with nitrogen gas. (DO≤1 ppm).    -   2. Glycerol was purge with nitrogen gas. (DO≤1 ppm). Sodium        chloride (Example 10) was added to glycerol and stirred until it        is completely dissolved.    -   3. 70% of nitrogen purged Absolute Ethanol was taken.    -   4. Cyclophosphamide was added to step-3 and dissolved by        stirring at 1000 rpm for 15 minutes i.e. until clear solution        forms under continuous nitrogen purging.    -   5. Step-4 was added to step-2 and mixed well for uniform        distribution.    -   6. The volume was made to 100% using nitrogen purged Absolute        Ethanol and stir for 10 min for uniform distribution.    -   7. The bulk solution was filled in 2 ml glass vial and blanketed        with nitrogen in head space and stoppered with 13 mm stopper and        seal with 13 mm seal.

Exam- Exam- Exam- Exam- ple 14 ple 15 ple 16 ple 17 IngredientsQuantity/mL Cyclophosphamide 500 mg 500 mg 500 mg 500 mg Polyethylene0.1 mL to — — — glycol (PEG300) 0.9 mL Propylene glycol — 0.1 mL to — —0.9 mL Polysorbate 80 — — 0.1 mL to — 0.9 mL Cremophor EL — — — 0.1 mLto 0.9 mL Anhydrous Qs to Qs to Qs to Qs to Ethanol 1 mL 1 mL 1 mL 1 mLManufacturing Process:

-   1. Anhydrous ethanol was purged with nitrogen gas. (DO≤1 ppm).-   2. Cyclophosphamide was dissolved in 100% PEG 300 (example 11) or    propylene glycol (example 12), polysorbate 80 (example 13) or    cremophor EL (Example 14) and dissolved by stirring at 1000 rpm for    15 minutes i.e. until clear solution forms under continuous nitrogen    purging.-   3. Volume make up done with pre-purged anhydrous ethanol to 100%.-   4. Bulk solution was purged with nitrogen to remove the dissolve    oxygen content to minimum (preferably DO≤1 ppm).-   5. The bulk solution was filled in 2 ml glass vial and blanketed    with nitrogen in head space and stoppered with 13 mm stopper and    seal with 13 mm seal.

The invention claimed is:
 1. A stable liquid pharmaceutical formulationof cyclophosphamide comprising: cyclophosphamide; and at least onepharmaceutically acceptable excipient, the liquid pharmaceuticalformulation having moisture content of less than about 2.0% by weight,wherein the liquid pharmaceutical formulation is stable when themoisture content is reduced to less than about 2.0% by weight; whereintotal impurities are less than about 6.0% by weight when the liquidpharmaceutical formulation is stored at 2-8° C. for at least six months;and wherein said excipient excludes polyol organic solvent and citricacid.
 2. The stable liquid formulation of claim 1, wherein the moisturecontent is less than about 1.0% by weight.
 3. The stable liquidformulation of claim 1, wherein the concentration of cyclophosphamide isat least 0.1 g per mL.
 4. The stable liquid formulation of claim 1,wherein the concentration of the cyclophosphamide is 0.5 g per mL. 5.The stable liquid formulation of claim 1, wherein said liquidformulation has less than about 1.5% of impurity D, represented by thestructure IV


6. The stable liquid formulation of claim 1, wherein said liquidformulation has less than about 1.5% of impurity B, represented by thestructure III


7. The stable liquid formulation of claim 1 wherein the cyclophosphamidein the formulation is cyclophosphamide monohydrate.
 8. The stable liquidformulation of claim 1 wherein the pharmaceutically acceptable excipientis selected from the group comprising of ethanol, polysorbates,cyclodextrins, dimethyl acetamide, polyethoxylate castor oil orcombination thereof.
 9. The stable liquid formulation of claim 1 whereintotal impurities are less than about 6.0% by weight when the liquidpharmaceutical formulation is stored at 2-8° C. for six months.